CN102062735B - Biomarker diagnostic kit for acute coronary syndrome - Google Patents
Biomarker diagnostic kit for acute coronary syndrome Download PDFInfo
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Abstract
The invention discloses a biomarker detection method and a diagnostic kit for acute coronary syndrome. The detection method is characterized in that: a plurality of biomarkers in the same sample can be detected once, namely, a biotin-marked detection antibody-acute coronary syndrome biomarker-capture antibody-bead tetragenous complex is formed by the preparation of a liquid chip, and is combined with streptavidin-phycoerythrin (PE) to detect fluorescence signals of different beads, thereby determining the existence of different acute coronary syndrome biomarkers in the sample to be detected and the acute coronary syndrome biomarker content of the sample. The invention also discloses components of the diagnostic kit. The method and the kit provided by the invention have the advantages of high sensitivity, high flux, high detection speed, detection accuracy and the like, and can simultaneously realize the qualitative and quantitative detection of the plurality of acute coronary syndromebiomarkers.
Description
Technical field
The present invention relates to a kind of in-vitro diagnosis detection method and diagnostic kit, particularly relate to liquid-phase chip combined parallel detecting method and the diagnostic kit thereof of the biomarker of various acute coronary syndrome.
Background technology
Acute coronary syndrome (Acute Coronary Syndrome, ACS) be to be decided to be the basic pathology physilogical characteristics with the Coronary Atherosclerotic Plaque shakiness, take acute myocardial ischemia as common trait, one group of clinical syndrome take pectoralgia as classical symptom.ACS is the main cause of the medical and sudden death of most of cardiovascular patients clinically, the serious harm mankind's life and health.The biomarker of ACS (following can referred to as " ACS mark ") not only can the Accurate Diagnosis acute coronary syndrome, estimates its order of severity, can also guiding clinical treatment and the monitoring of curative effect, judge the prognosis of acute coronary syndrome.Therefore the associating parallel detection of various dissimilar ACS marks has become inevitablely, and the rapid property of the high flux of many indexs parallel detection, Stability and veracity are just most important.
CK-MB (CK-MB) is considered to diagnose " goldstandard " of ACS because tissue specificity is good always.The biomarker relevant with ACS that Recent study is found also comprises: serum cardiac troponin T (cTnT), cardiac muscle troponin I (cTnI), creatine kinase isozyme (CK-MB), H-FABP (H-FABP), myoglobins (Myo), myeloperoxidase (MPO) etc.
At present, for the existing multiple detection method of the mensuration of ACS mark, comprise immunofluorescence analysis, enzyme-linked immuno assay (ELISA), radiommunoassay (RIA), the separation enzyme-linked immunoassay of magnetic (EIMA) etc.But these technology once can only detect for a kind of mark, and complex operation, sensitivity are relatively poor, can not really satisfy the needs that clinical diagnosis detects.Repeatability is poor, insufficient sensitivity good and the shortcoming of complex operation and the solid phase biological chip technology exists.
Liquid-phase chip technology (xMAP) is a kind of biochip technology platform that is widely used in the multiple biological respinses such as protein, gene, receptor/ligand, mainly comprises microballoon, probe molecule, detected material and four kinds of compositions of reporter molecules.In the middle of the manufacture process of microballoon, mixed two kinds of different redness classification fluorescence, different according to the ratio of these two kinds of fluorescence, sphere matrix is divided into 100 kinds, 100 kinds of different probe molecules on can mark, can be simultaneously in the sample nearly 100 kinds of different target molecules detect.In the course of reaction, probe and reporter molecules all respectively with the target molecule specific binding.After reaction finishes, make single microballoon by sense channel, use red, green two-color laser simultaneously the green report fluorescence on the classification fluorescence of the redness on the microballoon and the reporter molecules to be detected, can determine kind and the quantity of the detection thing of institute's combination.
The present invention is based on the outstanding advantages such as high sensitivity, high flux, the detection of liquid-phase chip technology be rapid, the multiple biomarker of ACS is carried out parallel detection, can better be used in clinical detection.
Summary of the invention
Technical matters to be solved by this invention provides a kind of liquid-phase chip combined parallel detecting method and diagnostic kit thereof for the acute coronary syndrome biomarker, this detection method and kit comprise the associating parallel detection for cTnT, cTnI, CK-MB, H-FABP, Myo, six kinds of ACS marks of MPO, have the advantages such as high sensitivity, high specific, good stability, detection be rapid.
For solving the problems of the technologies described above, the liquid-phase chip combined parallel detecting method of a kind of acute coronary syndrome biomarker of the present invention may further comprise the steps:
(1) with difference microballoon (Beads numbering, surperficial carboxyl modified, the numbering be respectively 11,15,21,33,35,37) activation after, make corresponding capture antibody (antibody of anti-ACS mark) and corresponding microballoon coupling, form " capture antibody-microballoon " bigeminy complex, described each capture antibody is anti-a kind of ACS mark respectively, thereby makes ACS mark and capture antibody in the testing sample form " ACS mark-capture antibody-microballoon " three complexs;
(2) different detection antibody is carried out biotin (Biotin) mark, wherein said each detect respectively anti-a kind of ACS mark and corresponding to capture antibody of antibody, and be incorporated into respectively the different epitopes of this mark from capture antibody;
(3) three complexs that step (1) formed mix with the biotin labeled detection antibody that contains in the step (2), thereby form " biotin labeled detection antibody-ACS biomarker-capture antibody-microballoon " tetrad complex;
(4) with after the tetrad complex in the step (3) and SA-PE (Streptavidin-PE) combination, detect the fluorescence signal of different microballoons, thereby determine existence and the content of various ACS marks in the detected sample.
The above detection method also comprises step: detected fluorescence signal and the typical curve measured in the step (4) compared, thus the content of various ACS marks in definite detected sample.
Described microballoon is that mean diameter is 5.6 μ m, and combines the polystyrene microsphere of different fluorescent dyes, i.e. color-code microballoon (color-coded beads).
Microballoon activation in the described step (1) refers to that microballoon activates in 3 mixed liquors that are made of 1-ethyl-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) solution, N-hydroxy thiosuccinimide (S-NHS) solution and activation damping fluid, wherein, the amount ratio of 3 mixed liquors is: when microballoon quantity is 2.5 * 10
6When individual, need 50mg/mlEDC solution 10 μ l: 50mg/mlS-NHS solution 10 μ l: 100mM NaH
2PO
4, pH6.3 activation damping fluid 80 μ l, the consumption of mixed liquor can be adjusted accordingly according to the quantity of microballoon.
Described capture antibody and detect antibody be for following 6 kinds can independent assortment the ACS mark capture antibody and detect antibody:
cTnT，cTnI，CK-MB，H-FABP，Myo，MPO。
Detects with Luminex (xMAP) in the step described in the detection method (4), the described fluorescence signal that detects is redness on the microballoon that excites of the red laser report fluorescence signal that phycoerythrin that fluorescence signal and green laser excite produces of classifying.
The problem of another solution of the present invention is, a kind of diagnostic kit that detects various acute coronary syndrome biomarker is provided, and mainly comprises following component:
(1) microballoon of coupling antibody: contain 6 kinds of couplings the carboxyl microballoon of capture antibody (microballoon is numbered 11,15,21,33,35,37), namely be respectively: coupling the microballoon 11 of cTnT capture antibody, coupling the microballoon 15 of cTnI capture antibody, coupling the microballoon 21 of CK-MB capture antibody, coupling the microballoon 33 of H-FABP capture antibody, coupling the microballoon 35 of Myo capture antibody, coupling the microballoon 37 of MPO capture antibody, every kind of capture antibody is anti-a kind of ACS mark and be coupled to the microballoons of different numberings respectively, forms the bigeminy complex of " capture antibody-microballoon ";
(2) biotin labeled detection antibody: contain biotin labeled cTnT and detect antibody, biotin labeled cTnI detects antibody, biotin labeled CK-MB detects antibody, contain biotin labeled H-FABP and detect antibody, contain biotin labeled Myo and detect antibody, contain biotin labeled MPO and detect antibody, wherein, described each detect respectively anti-a kind of corresponding ACS mark and corresponding to capture antibody of antibody, and be incorporated into respectively the different epitopes of this biomarker from capture antibody;
(3) SA-PE (Streptavidin-PE): wherein Streptavidin can with the biotin specific binding, form the fluorescein-labeled detection antibody of band phycoerythrin (PE), the green laser by the liquid-phase chip instrument excites phycoerythrin to carry out fluoroscopic examination;
(4) standard items: the standard items that comprise the biomarker (antigen) of various ACS;
(5) quality-control product: comprise positive control and negative control.
Wherein said capture antibody and detect antibody be for following 6 kinds can independent assortment the ACS mark capture antibody and detect antibody:
cTnT，cTnI，CK-MB，H-FABP，Myo，MPO。
A kind of diagnostic kit that detects the acute coronary syndrome biomarker of the present invention can be used for detecting existence and the content of ACS mark in the vitro samples, and the diagnosis that also can be used for other cardiovascular and cerebrovascular diseases except ACS detects or prediction.
Because the present invention has utilized liquid-phase chip technology, make detection method and kit have the outstanding advantages such as high sensitivity, high specific, high flux, good stability, detection be rapid, accurate, can carry out simultaneously to the biomarker of various acute coronary syndrome quantitative and qualitative analysis and detect, can better be used in clinical detection.
Description of drawings
The present invention is further detailed explanation below in conjunction with accompanying drawing and embodiment:
Fig. 1 is the concentration profile that detects cTnT in ACS patient and the Normal group among the present invention;
Fig. 2 is the concentration profile that detects cTnI in ACS patient and the Normal group among the present invention;
Fig. 3 is the concentration profile that detects CK-MB in ACS patient and the Normal group among the present invention;
Fig. 4 is the concentration profile that detects H-FABP in ACS patient and the Normal group among the present invention;
Fig. 5 is the concentration profile that detects Myo in ACS patient and the Normal group among the present invention;
Fig. 6 is the concentration profile that detects MPO in ACS patient and the Normal group among the present invention.
Embodiment
Experiment material:
6 kinds of ACS marks (antigen) and corresponding antibodies that the present invention is used derive from Biodesign, cellsciences and Abcam company;
Microballoon (surperficial carboxyl modified), the SA-PE of different numberings are all purchased the company in QIAGEN;
1-ethyl-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC), N-hydroxy thiosuccinimide (S-NHS) and N-hydroxy thiosuccinimide biotin (S-NHS-Biotin) are purchased the company in Pierce.
The damping fluid preparation:
Activation damping fluid (Activation buffer): 100mM NaH
2PO
4, pH6.3;
Coupling buffer (Coupling buffer): 50mM HEPES, pH7.4;
Phosphate buffer (PBS): 10mM NaH
2PO
4, 150mM NaCl, pH7.4.
The liquid-phase chip combined parallel detecting method of embodiment 1:3 kind acute coronary syndrome mark
Concrete detection method comprises the steps:
1. the activation of required microballoon:
1.1 full speed vortex microballoon storage liquid is 3min at least, forms the microballoon suspension of homogeneous;
1.2 take by weighing respectively among the EDC and S-NHS to two centrifuge tube of 10mg;
1.3 making its final concentration with deionized water dissolving is 50mg/ml;
1.4 get the centrifugal 3min of microballoon suspension 10000g of 1ml, carefully remove supernatant;
1.5 it is resuspended with microballoon to add the activation damping fluid of 80 μ l;
1.6 add respectively the EDC solution (50mg/ml) of 10 μ l and the S-NHS solution (50mg/ml) of 10 μ l, mix room temperature (15-25 ℃), lucifuge, oscillation incubation 20min.
2. the microballoon coupling of corresponding capture antibody and activation
2.1 with coupling buffer capture antibody being diluted to volume is 500 μ l, concentration is the solution of 0.1mg/ml; (can not contain foreign protein in the antibody-solutions, azide, aminoacetic acid, Tris or other any reagent that contains amino.If contain these reagent, remove by dialysis or gel permeation chromatography.)
2.2 microballoon at the centrifugal 3min of 10000g, is carefully removed supernatant;
2.3 add and diluted good antibody-solutions (500 μ l) in the step 2.1;
2.4 with microballoon and the antibody-solutions of activation, under room temperature (15-25 ℃), lucifuge, oscillation incubation 2h; (centrifuge tube must wrap up lucifuge with tinfoil)
2.5 microballoon at the centrifugal 3min of 10000g, is carefully removed supernatant;
2.6 it is resuspended with microballoon to add 500 μ l PBS, the centrifugal 3min of 10000g carefully removes supernatant;
2.7 add 1ml PBS/1%BSA (BSA: bovine serum albumin(BSA)) that microballoon is resuspended;
2.8 by thrombocytometer microballoon is counted;
3. biotin labeling detects antibody
3.1 biotin reagent (S-NHS-Biotin) is taken out from the reefer of refrigerator, and at room temperature balance makes it return to room temperature;
3.2 according to the concentration that detects antibody, with PBS antibody dilution is arrived 1mg/ml;
If being dissolved in, antibody contains in the amino solution, should be first except deaminizing, and being replaced as does not have amino damping fluid;
3.3 the biotin solution with ultrapure water configuration 10mM;
3.4 1: 20 in molar ratio (antibody: biotin), calculate the needed amount of biotin, join in the antibody-solutions that concentration is 1mg/ml;
Computing formula:
3.5 reactant liquor is hatched 2h on ice, or at incubated at room 30min;
3.6 reactant liquor is transferred to dialysis cassette, removes wherein unreacted S-NHS-Biotin.
4. the configuration of antigen standard items
CTnT prepares by the concentration of 31.25,6.25,1.25,0.25,0.05,0.01,0ng/ml, Myo and MPO prepare by the concentration of 3125,625,125,25,5,1,0ng/ml, the mark mixed liquor is labeled as respectively STD6, STD5, STD4, STD3, STD2, STD1, STD0.
5. the preparation of the microballoon mixed liquor of coupling capture antibody (I mixed liquor)
The microballoon of capture antibody of 3 kinds of ACS marks of having got coupling respectively is as following: cTnT capture antibody microballoon 11, Myo capture antibody microballoon 35, MPO capture antibody microballoon 37, equal proportion is mixed, and makes the final concentration of every kind of microballoon be respectively 200/μ l, and 4 ℃ keep in Dark Place.
6. contain the preparation of biotin labeled detection antibody mixed liquor (II mixed liquor)
Get respectively and carried out biotin labeled cTnT detection antibody, Myo detects antibody, and MPO detects antibody, adds the PBS of pH7.4, makes every kind of final concentration that detects antibody be respectively 10 μ g/ml.
7. quality-control product
Quality-control product comprises positive control and negative control.
8. the content detection of 3 kinds of ACS biomarkers in the blood serum sample
8.1 blood serum sample comprises 12 parts in normal human serum sample, 12 parts in ACS patients serum sample.
8.2 add respectively the microballoon mixed liquor (I mixed liquor) of coupling capture antibody in 96 hole ELISA Plate, 25 μ l/ holes;
8.3 add standard items (STD0, STD1, STD2, STD3, STD4, STD5, STD6), quality-control product (positive control and negative control), patients serum's sample 1-12 number, normal human serum sample 1-12 number, 25 μ l/ holes;
8.4 with the up and down mixing potpourri of volley of rifle fire tenderness, cover lid, at room temperature lucifuge is hatched 30min;
Contain biotin labeled detection antibody mixed liquor (II mixed liquor), 25 μ l/ holes 8.5 add; With the up and down mixing potpourri of volley of rifle fire tenderness, cover lid, at room temperature lucifuge is hatched 30min;
8.6 with PBS/1%BSA SA-PE is diluted to the solution that concentration is 200 μ g/ml, add the good Streptavidin phycoerythrin 25 μ l of dilution in each hole, with the up and down mixing potpourri of volley of rifle fire tenderness, cover lid, at room temperature lucifuge is hatched 15-30min;
8.7 on liquid-phase chip instrument (LiquiChip 200, QIAGEN company), the potpourri of reaction is analyzed, automatic drawing standard curve, and calculate the content of 3 kinds of ACS marks in the test sample according to typical curve.
8.8 testing result and analysis
Specifically referring to table 1-3.
The concentration value of table 1ACS group biomarker
| The ACS group | cTnT (ng/ml) | Myo (ng/ml) | MPO (ng/ml) |
| 1 | 0.73 | 217 | 103 |
| 2 | 1.16 | 459 | 142 |
| 3 | 0.54 | 285 | 87 |
| 4 | 0.81 | 326 | 95 |
| 5 | 1.07 | 548 | 151 |
| 6 | 0.62 | 173 | 86 |
| 7 | 0.95 | 362 | 110 |
| 8 | 0.78 | 294 | 98 |
| 9 | 0.69 | 380 | 102 |
| 10 | 0.92 | 436 | 129 |
| 11 | 0.75 | 269 | 93 |
| 12 | 0.84 | 352 | 117 |
| Mean value | 0.82 | 341.75 | 109.42 |
The concentration value of table 2 Normal group biomarker
| Normal group | cTnT (ng/ml) | Myo (ng/ml) | MPO (ng/ml) |
| 1 | 0.09 | 38 | 45 |
| 2 | 0.06 | 25 | 58 |
| 3 | 0.11 | 46 | 36 |
| 4 | 0.07 | 31 | 50 |
| 5 | 0.10 | 42 | 55 |
| 6 | 0.08 | 23 | 34 |
| 7 | 0.06 | 17 | 41 |
| 8 | 0.09 | 34 | 43 |
| 9 | 0.12 | 49 | 47 |
| 10 | 0.11 | 37 | 53 |
| 11 | 0.07 | 24 | 38 |
| 12 | 0.10 | 41 | 52 |
| Mean value | 0.09 | 33.92 | 46 |
The biomarker mean value contrast of table 3ACS patient and normal group
| Myocardial injury markers | cTnT (ng/ml) | Myo (ng/ml) | MPO (ng/ml) |
| The ACS cell mean | 0.82 | 341.75 | 109.42 |
| The normal control cell mean | 0.09 | 33.92 | 46 |
| The ratio of the mean concentration of ACS group and Normal group | 9.11 | 10.08 | 2.38 |
Above result shows, can carry out the associating parallel detection to 3 kinds of ACS marks simultaneously with the inventive method, the concentration mean value of 3 kinds of marks can be found out from table 1-3,3 kinds of biomarkers of this of ACS patient, the cTnT group exceeds 9.11 times of Normal groups, and the Myo group exceeds 10.08 times of Normal groups, and the MPO group exceeds 2.38 times of Normal groups, have significant difference, can be used as the important indicator that clinical diagnosis in heart failure detects.
The concrete detection method of liquid-phase chip combined parallel detecting method of embodiment 2:6 kind acute coronary syndrome mark, the 1-3 step is with embodiment 1.
4. the configuration of antigen standard items
CTnT, cTnI are by 31.25,6.25,1.25,0.25,0.05,0.01 the concentration of 0ng/ml is prepared, CK-MB and H-FABP are by 312.5,62.5,12.5,2.5 the concentration of 0.5,0.1,0ng/ml is prepared, Myo and MPO are by 3125,625,125,25,5,1, the concentration of 0ng/ml is prepared, and the mark mixed liquor is labeled as respectively STD6, STD5, STD4, STD3, STD2, STD1, STD0.
5. the preparation of the microballoon mixed liquor of coupling capture antibody (I mixed liquor)
Get respectively the microballoon of the capture antibody that has been coated with 6 kinds of ACS marks, as following: cTnT capture antibody microballoon 11, cTnI capture antibody microballoon 15, CK-MB capture antibody microballoon 21, H-FABP capture antibody microballoon 33, Myo capture antibody microballoon 35, MPO capture antibody microballoon 37, equal proportion is mixed, and makes the final concentration of every kind of microballoon be respectively 200/μ l, and 4 ℃ keep in Dark Place.
6. contain the preparation of biotin labeled detection antibody mixed liquor (II mixed liquor)
Get respectively and carried out biotin labeled cTnT detection antibody, cTnI detects antibody, and CK-MB detects antibody, and H-FABP detects antibody, and Myo detects antibody, and MPO detects antibody, adds the PBS of pH7.4, makes every kind of final concentration that detects antibody be respectively 10 μ g/ml.
7. reference substance
Reference substance comprises positive control and negative control.
8. the content detection of 6 kinds of ACS biomarkers in the blood serum sample
8.1 blood serum sample comprises 26 parts in normal human serum sample, 26 parts in ACS patients serum sample.
8.2 add respectively the microballoon mixed liquor (I mixed liquor) of coupling capture antibody in 96 hole ELISA Plate, 25 μ l/ holes;
8.3 add standard items (STD0, STD1, STD2, STD3, STD4, STD5, STD6), quality-control product (positive control and negative control), patients serum's sample 1-26 number, normal human serum sample 1-26 number, 25 μ l/ holes;
8.4 with the up and down mixing potpourri of volley of rifle fire tenderness, cover lid, at room temperature lucifuge is hatched 30min;
Contain biotin labeled detection antibody mixed liquor (II mixed liquor), 25 μ l/ holes 8.5 add; With the up and down mixing potpourri of volley of rifle fire tenderness, cover lid, at room temperature lucifuge is hatched 30min;
8.6 with PBS/1%BSA SA-PE is diluted to the solution that concentration is 200 μ g/ml, add the good Streptavidin phycoerythrin 25 μ l of dilution in each hole, with the up and down mixing potpourri of volley of rifle fire tenderness, cover lid, at room temperature lucifuge is hatched 15-30min;
8.7 on liquid-phase chip instrument (LiquiChip 200, QIAGEN company), the potpourri of reaction is analyzed, automatic drawing standard curve, and calculate the content of 6 kinds of ACS biomarkers in the test sample according to typical curve.
8.8 testing result and analysis
Referring to table 4-6 and Fig. 1-5.
Table 4ACS group biomarker concentration value
| The ACS group | cTnT (ng/ml) | cTnI (ng/ml) | CK-MB (ng/ml) | H-FABP (ng/ml) | Myo (ng/ml) | MPO (ng/ml) |
| 1 | 0.68 | 1.25 | 13.6 | 32 | 203 | 117 |
| 2 | 1.04 | 2.47 | 20.1 | 76 | 396 | 145 |
| 3 | 0.57 | 1.09 | 8.9 | 49 | 251 | 92 |
| 4 | 0.76 | 1.14 | 11.8 | 58 | 287 | 106 |
| 5 | 1.10 | 2.31 | 17.0 | 80 | 472 | 148 |
| 6 | 0.59 | 0.86 | 9.5 | 34 | 183 | 90 |
| 7 | 0.91 | 2.18 | 19.2 | 61 | 314 | 123 |
| 8 | 0.67 | 1.42 | 12.4 | 53 | 269 | 104 |
| 9 | 0.55 | 1.03 | 9.3 | 65 | 326 | 115 |
| 10 | 0.86 | 2.16 | 23.9 | 71 | 348 | 131 |
| 11 | 0.73 | 1.59 | 14.7 | 46 | 230 | 89 |
| 12 | 0.82 | 1.84 | 16.8 | 54 | 315 | 126 |
| 13 | 1.15 | 2.68 | 22.6 | 86 | 457 | 143 |
| 14 | 0.64 | 1.20 | 9.0 | 30 | 214 | 112 |
| 15 | 0.93 | 2.07 | 18.1 | 83 | 460 | 139 |
| 16 | 0.79 | 1.71 | 14.5 | 67 | 363 | 124 |
| 17 | 0.61 | 0.93 | 8.2 | 42 | 242 | 97 |
| 18 | 0.87 | 1.64 | 12.7 | 69 | 359 | 132 |
| 19 | 1.02 | 2.56 | 24.3 | 75 | 428 | 129 |
| 20 | 0.90 | 1.98 | 14.9 | 78 | 377 | 135 |
| 21 | 0.75 | 1.40 | 10.1 | 51 | 271 | 118 |
| 22 | 0.71 | 1.32 | 11.6 | 56 | 285 | 103 |
| 23 | 0.66 | 0.95 | 7.2 | 28 | 195 | 96 |
| 24 | 0.89 | 2.23 | 16.5 | 62 | 304 | 120 |
| 25 | 0.97 | 2.11 | 15.8 | 74 | 436 | 127 |
| 26 | 1.08 | 1.92 | 17.4 | 57 | 229 | 108 |
| Mean value | 0.82 | 1.69 | 14.62 | 59.12 | 315.92 | 118.04 |
Table 5 Normal group biomarker concentration value
| Normal group | cTnT (ng/ml) | cTnI (ng/ml) | CK-MB (ng/ml) | H-FABP (ng/ml) | Myo (ng/ml) | MPO (ng/ml) |
| 1 | 0.08 | 0.11 | 1.1 | 0.9 | 31 | 53 |
| 2 | 0.06 | 0.14 | 2.8 | 2.2 | 20 | 61 |
| 3 | 0.10 | 0.11 | 0.5 | 1.0 | 39 | 34 |
| 4 | 0.08 | 0.12 | 0.8 | 1.5 | 28 | 57 |
| 5 | 0.09 | 0.15 | 1.3 | 2.4 | 36 | 60 |
| 6 | 0.07 | 0.08 | 0.6 | 0.7 | 18 | 39 |
| 7 | 0.05 | 0.12 | 2.3 | 1.8 | 24 | 48 |
| 8 | 0.09 | 0.10 | 1.1 | 1.4 | 26 | 52 |
| 9 | 0.10 | 0.08 | 0.7 | 1.9 | 45 | 46 |
| 10 | 0.11 | 0.13 | 1.9 | 2.0 | 32 | 51 |
| 11 | 0.06 | 0.10 | 1.0 | 0.9 | 21 | 35 |
| 12 | 0.08 | 0.12 | 1.5 | 1.6 | 37 | 49 |
| 13 | 0.05 | 0.14 | 2.1 | 2.5 | 43 | 54 |
| 14 | 0.11 | 0.10 | 0.6 | 1.1 | 29 | 37 |
| 15 | 0.07 | 0.14 | 1.9 | 2.6 | 40 | 50 |
| 16 | 0.09 | 0.11 | 1.3 | 1.8 | 35 | 41 |
| 17 | 0.06 | 0.09 | 0.4 | 0.6 | 23 | 34 |
| 18 | 0.08 | 0.11 | 0.9 | 2.1 | 34 | 45 |
| 19 | 0.10 | 0.15 | 2.6 | 2.4 | 41 | 56 |
| 20 | 0.09 | 0.13 | 1.2 | 2.2 | 38 | 42 |
| 21 | 0.07 | 0.12 | 0.8 | 1.3 | 27 | 33 |
| 22 | 0.08 | 0.09 | 1.0 | 1.5 | 30 | 47 |
| 23 | 0.06 | 0.07 | 0.5 | 0.7 | 22 | 36 |
| 24 | 0.09 | 0.13 | 1.7 | 1.9 | 33 | 43 |
| 25 | 0.07 | 0.12 | 1.6 | 2.5 | 21 | 38 |
| 26 | 0.10 | 0.13 | 1.5 | 1.6 | 39 | 55 |
| Mean value | 0.08 | 0.12 | 1.30 | 1.66 | 31.23 | 46 |
The myocardial injury markers mean value contrast of table 6ACS patient and normal group
| Myocardial injury markers | cTnT (ng/ml) | cTnI (ng/ml) | CK-MB (ng/ml) | H-FABP (ng/ml) | Myo (ng/ml) | MPO (ng/ml) |
| ACS organizes average group | 0.82 | 1.69 | 14.62 | 59.12 | 315.92 | 118.04 |
| The normal control cell mean | 0.08 | 0.12 | 1.30 | 1.66 | 31.23 | 46 |
| The ratio of the mean concentration of ACS group and Normal group | 10.25 | 14.08 | 11.25 | 35.61 | 10.12 | 2.57 |
Above result shows, can carry out the associating parallel detection to 6 kinds of ACS marks simultaneously with the inventive method, but also can carry out according to the actual requirements the joint-detection of various combination to 6 kinds of ACS marks.The concentration mean value of 6 kinds of marks can be found out from table 4-6,6 kinds of biomarkers of this of ACS patient, wherein the MPO group exceeds 2.5 times of Normal groups, other mark groups all exceed more than 10 times, wherein the H-FABP group can reach 35.61 times, have significant difference, can be used as the important indicator that the ACS clinical diagnosis detects.
About after the those set forth of the present invention, those skilled in the art can do various modifications or variation to the present invention more than having read, and these equivalent form of values belong to the scope defined in the application's appended claims equally.
Claims (2)
1. a diagnostic kit that detects various acute coronary syndrome biomarker is characterized in that, comprises following chief component composition:
(1) microballoon of coupling antibody: the 6 kinds of microballoons of different capture antibodies that contained respectively coupling, be respectively: coupling the microballoon of serum cardiac troponin T cTnT capture antibody, coupling the microballoon of cardiac muscle troponin I cTnI capture antibody, coupling the microballoon of creatine kinase isozyme CK-MB capture antibody, coupling the microballoon of H-FABP H-FABP capture antibody, coupling the microballoon of myoglobins Myo capture antibody, coupling the microballoon of myeloperoxidase MPO capture antibody, every kind of capture antibody resists respectively a kind of acute coronary syndrome biomarker and is coupled to different microballoons of numbering, and forms the bigeminy complex of " antibody-microballoon ";
(2) biotin labeled detection antibody: contain biotin labeled serum cardiac troponin T cTnT and detect antibody, contain biotin labeled cardiac muscle troponin I cTnI and detect antibody, biotin labeled creatine kinase isozyme CK-MB detects antibody, biotin labeled H-FABP H-FABP detects antibody, biotin labeled myoglobins Myo detects antibody, biotin labeled myeloperoxidase MPO detects antibody, wherein said each detect respectively anti-a kind of corresponding acute coronary syndrome biomarker and corresponding to capture antibody of antibody, and be incorporated into respectively the different epitopes of this biomarker from capture antibody;
(3) SA-PE Streptavidin-PE: wherein Streptavidin can with the biotin specific binding, form the fluorescein-labeled detection antibody of band phycoerythrin;
(4) standard items: the standard items that comprise the biomarker of various acute coronary syndromes;
(5) quality-control product: comprise positive control and negative control;
Described microballoon is a kind of color-code microballoon color-coded beads, and mean diameter is 5.6 μ m, and combines the polystyrene microsphere of the surperficial carboxyl modified of different fluorescent dyes.
2. the diagnostic kit of detection various acute coronary syndrome biomarker as claimed in claim 1, it is characterized in that, described capture antibody and detection antibody are for the capture antibody of following 6 kinds of acute coronary syndrome biomarkers and detect antibody:
Serum cardiac troponin T cTnT, cardiac muscle troponin I cTnI, creatine kinase isozyme CK-MB, H-FABP H-FABP, myoglobins Myo, myeloperoxidase MPO.
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